Alkalinization Transients in Individual Mitochondria

Abstract

Listen

Translate article

The pH within the mitochondrial matrix (pHmito) is an important bioenergetic parameter well studied in isolated mitochondria but poorly characterized in intact cells, and all in situ studies available report spatially averaged measurements of pHmito. We used a new pH-sensitive GFP-based fluorescent probe targeted to the mitochondrial matrix, mito-SypHer, to study pHmito homeostasis at the level of single mitochondria in intact living cells. We observed that individual mitochondria undergo spontaneous alkalinization transients. The pH transients occurred randomly in time and space and had a characteristic profile, with a rapid onset (time to peak 1.6±0.1 sec), a slower decay (τ=8.5±0.6 sec), and an average amplitude of 0.38±0.05 pH units. The pH transients were absent in Rho-0 cells that lack a functional respiratory chain, were abrogated by protonophores, occurred concomitantly with transient mitochondrial depolarization events measured with TMRE, and their frequency was strongly decreased by respiratory chain inhibitors. These kinetics and functional properties resemble the “superoxide flashes” previously reported in single mitochondria with a pericam-derived probe. However, the fluorescence of purified mito-SypHer was not altered by all the ROS tested including superoxide, and in live cells increasing the pH buffering power of mitochondria with NH4Cl decreased the amplitude and slowed the kinetics of the transients, confirming that these were caused by protons. The pH spikes were not spatially restricted to single mitochondria but were also observed in clusters of interconnected mitochondria and their spatial extent was altered by enforced fusion or fission of mitochondria. Our data indicate that in live cells individual mitochondria undergo spontaneous basification transients that require functional OXPHOS machinery. The synchronicity between the spontaneous pH spikes and depolarization events suggests that H+ extrusion by the respiratory chain complexes or by the reverse mode of the ATP-synthase might be transiently enhanced during bouts of depolarization.